Evidence for the Existence of Two ^- Pools and Two Biosynthetic ^-Carotene Pathways in the Chloroplast K. H. Grumbach Botanisches Institut der Universität Karlsruhe, Kaiserstraße 12, D-7500 Karlsruhe

Z. Naturforsch. 34 c, 1205— 1208 (1979); received September 4, 1979

Biosynthesis, , /?-Carotene, Photosynthesis Two /7-Carotene pools were obtained in the chloroplast. It is concluded that one pool is very small like a-carotene and responsible exclusively for the biosynthesis of /?--. The other ^-carotene pool is proposed to be the one, that is located close to photosystem I and is involved in photosynthesis as a light protecting agent for chlorophylls against photo-oxidation. Furthermore evidence is given that both /7-carotene pools are synthesized by independent biosyn­ thetic pathways.

Introduction chloroplast in two different pools which are proba­ bly synthesized by independent biosynthetic path­ The intraorganelle distribution of chlorophylls, ways. carotenoids and quinones in the chloroplast is differ­ ent. Chlorophylls are present exclusively in the thy­ lakoids [!]• Besides chlorophylls, carotenoids and Materials and Methods quinones are constituents of the photosynthetic Chlorella pyrenoidosa (211-81, algae collection membrane, but they are also present in the osmio- Göttingen) was grown autotrophically for 7—10 philic plastoglobuli [2, 3]. Furthermore investiga­ days and then supplied with [4,5-3H] leucine tions carried out by Douce reveal, that carotenoids (500 |iCi, specific activity l.OmCi/mmol) for a pe­ () are contained even in the chloroplast riod of 18 h. During the final 40 min [2-14C] acetate envelope [4, 5]. (200 nCi, specific activity 55 mCi/mmol) was also Fractionation studies of chloroplast lamellae have supplied. Thereafter the cells were washed free from also obtained differences in the distribution of chlo­ exogenous labelled substrate and reincubated in the rophylls, carotenoids and quinones between photo­ light in fresh nutrition medium. At different times system I and II and the light-harvesting complex after the beginning of the experiment Chlorella cells [6 — 10]. Whereas /7-carotene and chlorophyll a are were harvested, carotenoids isolated, purified to a the major pigments of photosystem I-, chlorophyll a constant specific radioactivity and assayed for 14C- and are present in large amounts in photosys­ and 3H-radioactivity by liquid scintillation counting. tem II-particles together with plastoquinone-9, a-to- Radish seedlings grown for 6 days in light were in­ copherol and lower concentrations of /7-carotene, cubated with [2-3H]mevalonate (1 mCi, specific activ­ violaxanthin and . In contrast to photosys­ ity 100 — 500 mCi/mmol) together with [2-14C] ace­ tem I and II every present in the chloro­ tate (1 mCi, specific activity 40 — 60 mCi/mmol) for plast is contained in the light-harvesting complex to­ 24 h. Thereafter carotenoids were isolated, purified gether with chlorophyll a and b [10]. to a constant specific radioactivity and assayed for In this communication dual labelling experiments 14C- and 3H-radioactivity. with [3H]leucine/[14C] acetate and [3H] mevalonate/- [14C] acetate were achieved to obtain differences in the intraplastidic compartmentation of carotenoids Results in chloroplasts of green radish seedlings and Chlorel­ Besides leucine, C 02, acetate and mevalonate are la pyrenoidosa. The aim of these investigations was the most common precursors for carotenoid biosyn­ to indicate in vivo that /7-carotene is contained in the thesis in algae and higher plants. The first specific re­ action in biosynthesis leading to the for­ Reprint requests to Dr. K. H. Grumbach. mation of carotenoids involves the biosynthesis of 0341-0382/79/1200-1205 $01.00/0 from geranylgeranylpyrophosphate [11]. 1206 K. H. Grumbach • Compartmentation of yS-Carotene in the Chloroplast

Phytoene Phytoene \ thesized directly from a-carotene can also be seen \ Phytofluene after the pulse chase when the 14C- and ^-radioactiv­ 1 i ♦ f ity is lost in a-carotene and at the same time increases 5-Carotene 5-Carotene I 1 in lutein. On the other hand was labelled ♦ ♦ Neurosporene Neurosporene to a much higher extent by [3H] leucine and [14C] ace­ / \ / X tate than its precursor /7-carotene. During the last ot-Zeacarotene (3-Zeacarotene p -Z eac aro ten e Lyc 60 min of the experiment 14C-radioactivity decreases \ / I 1 \ / in /7-carotene and increases in zeaxanthin due to the / \ transformation of [14C-/7] carotene into [14C] zeaxan­ >C arotene y-Carotene H-Carotene thin. 3H-radioactivity, however, was not lost to the i 1 ♦ » same extent but increased in /7-carotene and zeaxan­ ot-Carotene ß -C aro ten e i thin as well. The effect that the [14C]label was lost in * 1 Lutein Zeaxanthin /7-carotene to a greater extent than was ^-radioactiv­ I ity, as well as the higher mol specific 3H- and 14C-ra- dioactivity of zeaxanthin compared to /7-carotene is » Violaxanthin in contrast to the previous assumption that a carote­ » noid is contained in the chloroplast in one pool. It Neoxanthin can only be explained by the existence of two /7-caro- Fig. 1. General scheme for carotenoid biosynthesis [12] tene pools. From this it is concluded, that one /7-ca- and the proposed independent /?-carotene biosynthesis. rotene pool is present in the chloroplast as a small biosynthetic pool like a-carotene. This pool has a higher mol specific radioactivity than zeaxanthin, Phytoene then is desaturated via phytofluene, C-ca- whereas the other pool seems to be larger with a rotene and neurosporene to lycopene (Fig. 1). Lyco­ lower specific radioactivity. pene finally cyclizes to X- or y-carotene. On the other Further evidence that /?-carotene is contained in hand the cyclization of neurosporene to ß- and a-zea- the chloroplast in two different pools was obtained carotene and finally to X- or y-carotene was propos­ by dual labelling experiments with [2-3H] mevalona- ed as an alternative reaction. A-carotenc cyclizes to te and [2-14C] acetate in chloroplasts of green radish a-carotene, whereas y-carotene froms both a- and /?- seedlings. Besides this it was also investigated, wheth­ carotene. By introduction of oxygen a-carotene er these two /?-carotene pools are synthesized in one froms lutein, while /7-carotene is the precursor of zea­ or two independent biosynthetic pathways. It is xanthin. known that carotenoid biosynthesis proceeds via ge- It is generally believed that all carotenoids are ranylgeranylpyrophosphate and different acyclic ca­ synthesized in one biosynthetic pathway in the chlo­ rotene precursors like phytoene, phytofluene and ly­ roplast stroma and thereafter attached to tne thyla­ copene that cyclizes to X- and y-carotene and finally koid membrane. If a carotenoid is synthesized via to a- or /7-carotene. If carotenoids are synthesized in one biosynthetic pathway and is contained in the one biosynthetic pathway, then [14C] acetate and chloroplast in only one pool, then, in a pulse-chase- [3H]mevalonate are incorporated in the acyclic caro­ labelling experiment, the label in a carotenoid mole­ tenoid precursors and also in and xantho- cule that is derived from a terpenoid precursor like [3H] leucine should follow the same kinetic as that of another e. g. [14C] acetate resulting in a constant Table I. Mol-specific radioactivity of radish carotenoids after 24 hours incorporation with [2-14C] acetate and [2-3H]- 3H/14C-ratio. mevalonic acid as precursors. The 3H- and 14C-labelling kinetic of a- and /7-caro- Cafotenpidi {14qDPM/n«n©l 3h / 14c tSfie and their produett kite« anti äe£*aitä)ta fir«* Chlorella pyrenoidosa are shown in Fig. 2. After 18 h incubation time with [3H] leucine all carotenoids lycopene 208 x 10s 310X105 0.67 a-carotene 154X105 0 - were labelled by tritium. Mol specific radioactivity lutein 4x 10s 0 - was higher in a-carotene than in lutein reflecting their /7-carotene 24 x 105 0.9 X 10s 26.67 precursor-product relationship. That lutein is syn­ violaxanthin 6 x10s 0 - K. H. Grumbach • Compartmentation of /7-Carotene in the Chloroplast 1207

dpm /jmilS ol S 6 ß - CAROTENE yumol 101 10 ' >> > o * T t 1 n uc i 5 i h 10 § 20 40 60 90 120 180 min tight dpm dpm LUTEIN dpm yumol jumol j j f c j 6 ZEAXANTHIN jumol MO5 10 : -4P = Mo6 3H ______, 3h _____

______- o Lrfi =i

J 4i h 10 s 10 *T ---- r “ 1--- 1------1------1------r f- s 0 20 40 60 90 120 180 min 20 40 60 90 120 180 min light light Fig. 2. Labelling kinetic of a- and /7-carotene and their biosynthetic products lutein and zeaxanthin from Chlorella pyrenoi- dosa in a tracer kinetic experiment with [4,5-3H] leucine and [2-14C] acetate as precursors.

phylls after 24 h incubation time. As shown in Ta­ Discussion ble I, [3H] mevalonate and [14C] acetate were incorpo­ Chlorophylls and carotenoids are involved in pho­ rated in lycopene and ^-carotene whereas a-carote- tosynthesis as light absorbing agents. Besides chloro­ ne, lutein and violaxanthin were labeled only by phylls, /7-carotene and lutein are the major chloro­ [3H] mevalonate. The highest mol specific radioactiv­ plast pigments [12]. Whereas the function of chloro­ ity was obtained in lycopene and the lowest in lutein phylls as light harvesting pigments in the reaction and violaxanthin. This is consistent with the first ex­ centers of photosystem I and II and the light-harvest­ periment. ing complex is known [10], only little information is However, the differences obtained in the label of present concerning the function of carotenoids. lycopene and /7-carotene compared to a-carotene, lu­ Carotenoids are involved in the activity of photosys­ tein and violaxanthin in this experiment are not tem I and II [10, 12] but also in the cy­ conclusive with the fact that all carotenoids are syn­ cle [13]. Particularly /?-carotene is proposed to func­ thesized in the chloroplast in one biosynthetic path­ tion as a light protecting agent for chlorophylls way. They rather support the existence of two /7-ca- against photooxidation by deactivating chlorophyll rotene pools in the chloroplast and also the existence triplets or singlet oxygen [14]. Furthermore it was of two independent biosynthetic pathways for the pointed out, that a large ^-carotene pool is associ­ forMstio» of l-6araieae. There seems to exist a sat all ated with the ante**» pigmeats ©f pfeotesystcm I /7-carotene pol that is responsible exclusively for the [15]. biosynthesis of /7-ionone xanthophylls. Besides this The dual labelling experiments performed in this pool a larger /7-carotene pool is evident. This large communication, concerning the analysis of the intra- /?-carotene pool could be synthesized by an indepen­ plastidic distribution of carotenoids are in agree­ dent terpenoid pathway. ment with previous investigations and reveal that ß- 1208 K. H. Grumbach • Compartmentation of ^-Carotene in the Chloroplast carotene is contained in the chloroplast in different of incubation implies that this large /7-carotene pool pools. It is concluded, that one /?-carotene pool is as of photosystem I is synthesized by an independent small as a-carotene. As a biosynthetic pool this /2-ca­ terpenoid pathway (Fig. 2). For these investigations rotene could be used for the biosynthesis of ß-iono- represent only a preliminary result, further experi­ ne-xanthophylls. Besides this biosynthetic pool an­ ments concerning the intraplastidic distribution of other larger /7-carotene pool is present in the chloro­ carotenoids are in progress. plast. This large pool could be the /7-carotene pool that is located close to photosystem I and protects Acknowledgements the chlorophylls against photo-oxidation [14, 15]. Financial support by Royal Society London and The total discrimination of [14C] acetate compared to Deutsche Forschungsgemeinschaft is gratefully ac­ [3H] mevalonate in all radish xanthophylls after 24 h knowledged.

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